Mohr L, Schauer J I, Boutin R H, Moradpour D, Wands J R
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown 02129, MA, USA.
Hepatology. 1999 Jan;29(1):82-9. doi: 10.1002/hep.510290148.
Gene therapy approaches for the treatment of malignant tumors will require high-level expression of therapeutic genes in tumors compared with normal tissues. This may be achieved either by targeted gene delivery to tumor cells or by the use of tumor-specific promoters. Here, we describe the use of a novel conjugate consisting of a tumor-reactive monoclonal antibody (mAb), designated AF-20, coupled to a DNA-binding cationic amphiphile, cholesteryl-spermine, for gene delivery to hepatocellular carcinoma (HCC) cells. The high-affinity mAb, AF-20, recognizes a rapidly internalized 180-kd cell-surface glycoprotein that is abundantly expressed on HCC and other human tumors. The AF-20 mAb and an isotype-matched control antibody (C7-57) were covalently coupled to cholesteryl-spermine. Binding and internalization of AF-20-cholesteryl-spermine was confirmed by fluorescence microscopy using fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody. Following transfection of FITC-labeled oligonucleotides and ethidium monoazide-labeled plasmid DNA, cellular uptake and intracellular localization of nucleic acids were examined by laser scanning confocal microscopy. Transfection of luciferase or beta-galactosidase reporter genes complexed to AF-20-cholesteryl-spermine resulted in high levels of gene expression in AF-20 antigen-positive tumor cells. Very low levels of gene expression were observed using the control compound (C7-57-cholesteryl-spermine), which does not recognize the AF-20 tumor antigen or when AF-20 antigen-negative NIH 3T3 cells were transfected with AF-20-cholesteryl-spermine. Thus, covalent coupling of the AF-20 mAb to cholesteryl-spermine generated a highly specific and efficient nonviral vector system for targeted gene delivery to AF-20 antigen-positive HCC cells.
与正常组织相比，用于治疗恶性肿瘤的基因治疗方法需要在肿瘤中实现治疗基因的高水平表达。这可以通过将基因靶向递送至肿瘤细胞或使用肿瘤特异性启动子来实现。在此，我们描述了一种新型偶联物的应用，该偶联物由肿瘤反应性单克隆抗体（mAb）AF-20与DNA结合阳离子两亲物胆固醇-精胺偶联而成，用于将基因递送至肝癌（HCC）细胞。高亲和力单克隆抗体AF-20识别一种快速内化的180-kd细胞表面糖蛋白，该糖蛋白在肝癌和其他人类肿瘤中大量表达。将AF-20单克隆抗体和同型对照抗体（C7-57）共价偶联至胆固醇-精胺。使用异硫氰酸荧光素（FITC）标记的抗小鼠IgG抗体通过荧光显微镜确认AF-20-胆固醇-精胺的结合和内化。在转染FITC标记的寡核苷酸和单叠氮溴化乙锭标记的质粒DNA后，通过激光扫描共聚焦显微镜检查核酸的细胞摄取和细胞内定位。与AF-20-胆固醇-精胺复合的荧光素酶或β-半乳糖苷酶报告基因的转染导致AF-20抗原阳性肿瘤细胞中高水平的基因表达。使用不识别AF-20肿瘤抗原的对照化合物（C7-57-胆固醇-精胺）或用AF-20-胆固醇-精胺转染AF-20抗原阴性的NIH 3T3细胞时，观察到极低水平的基因表达。因此，AF-20单克隆抗体与胆固醇-精胺的共价偶联产生了一种高度特异性和高效的非病毒载体系统，用于将基因靶向递送至AF-20抗原阳性的肝癌细胞。
